Reaction mass of [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid and Phosphonic acid, P-[(tetrahydro-2-hydroxy-2-oxido-4H-1,4,2-oxazaphosphorin-4-yl)methyl]-

Endpoint summary

Description of key information

The key acute oral toxicity study, conducted according to OECD Test Guideline 423 and in compliance with GLP, reports an acute oral LD50 value between 500-1000 mg/kg (equivalent to 250-500 mg/kg pure active acid substance as the test material was a 50% aqueous solution, see Note 1) (SafePpharm 2003a).

In the acute dermal toxicity study for HEBMP-xNa, conducted according to OECD Test Guideline 402 and in compliance with GLP, the LD50 value was concluded to be greater than 2246 mg/kg bodyweight (equivalent to 2000 mg active salt/kg bodyweight, equivalent to 1600 1580 mg/kg bw active acid, see Note 2) (Harlan Laboratories, 2012b).

The interpretation of results assumes that the water and the sodium ions do not contribute to toxicity observed in the studies, hence the doses have been adjusted to reinterpret the result in terms of the dose of phosphonate (molecular or anions) only, which is termed the “active acid equivalent”. See Notes 1 and 2 for conversions.

In accordance with Column 2 of REACH Annex VIII, the acute toxicity study via the inhalation route (required in Section 8.5.2) does not need to be conducted as reliable data via the oral and dermal routes are available.

Note 1: the calculation of results in terms of active acid equivalent was done as follows: LD50 500-1000 mg/kg which already takes into account the specific gravity of the substance, as corrected by the performing laboratory. The test material is said to be a 49% active acid HEBMP, therefore the LD50 500-1000 mg/kg * 49% is equivalent to 250-500 mg HEBMP acid/kg.

Note 2: the calculation of results in terms of active acid equivalent was done as follows: LD50 >2246 mg/kg body weight is equivalent to 2000 mg/ active ingredient/kg body weight due to the presence of 11% water, which was corrected by the performing laboratory. Assuming the study was conducted at neutral pH, this would be equivalent to a predominance of HEBMP-3Na based on pKa values. The result of 2000 mg HEBMP-3Na/kg has been converted into 2000 mg HEBMP acid/kg by using

The result has been corrected to the equivalent dose of active acid using a molecular weight conversions:

MW HEBMP acid (249.1 g/mol) / MW HEBMP-3Na (315.04 g/mol) = 0.79

[when taking the average of the Molecular Weights for the cyclic and linear forms of the substances]

Therefore 2000 mg HEBMP-3Na/kg * 0.79 = 1580 mg HEBMP acid/kg body weight

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.09.2003-13.10.2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, UK
- Age at study initiation: 8-12 weeks
- Fasting period before study: overnight and for 4 hours after dosing
- Housing: the animals were housed in groupd of 3 in suspended solid floor polypropylene cages furnished with woodflakes
- Diet: laboratory diet, ad libitum
- Water: ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: undiluted or in distilled water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30 mg/ml

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg

Doses:

300 mg/kg, 2000 mg/kg

No. of animals per sex per dose:

3F

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: The animals were observed for deaths or overt signs of toxicity 0.5, 1, 2 and 4 hours after dosing and subsequently once daily. individual body weights were reccorded prior to dosing and seven and fourteen days after treatment or at death.

- Necropsy of survivors performed: yes

- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: The gross pathological examination consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. the appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

500 - 1 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: An extrapolation to 100% pure substance would give 250 – 500 mg/kg, based on 50% aqueous solution of the multiconstituent (~50:50 linear:cyclic substance).

Mortality:

Two animals treated at a dose level of 2000 mg/kg were found dead or killed in extremis two hours or one day after dosing. There were no deaths at a dose level of 300 mg/kg.

Clinical signs:

other: Signs of systemic toxicity noted at a dose level of 2000 mg/kg were hunched posture, ataxia, decreased respiratory rate, gasping, laboured and noisy respiration, loss of righting reflex, piloerection, ptosis, pallor of the extremities and red/brown staini

Gross pathology:

Abnormalities noted at necropsy of the animal that was found dead two hours after dosing were haemorrhagic lungs, dark liver, dark kidneys, haemorrhage of the non-glandular region of the stomach and severe haemorrhage of the gastric mucosa and small and large intestines. Abnormalities noted at necropsy of the animal that was killed in extremis one day after dosing were haemorrhage of the gastric mucosa and gaseous stomach and small and large intestines. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Other findings:

None reported.

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

An acute oral LD50 value between 500-1000 mg/kg (equivalent to 250-500 mg/kg pure substance) was reported in a study carried out according to an appropriate OECD guideline and in compliance with GLP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

250 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 16 May 2012 and 30 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF), Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147, amended 26 June 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were eight to twelve weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.

The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of five, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

PREPARATION OF TEST ITEM:
The test item contains 10.95% water. For the purposes of the study the test item concentration was corrected for this. The test item was weighed out according to each animal's individual bodyweight and moistened with distilled water prior to application.
The absorption of the test item was not determined.

TEST SITE
On the day before treatment the back and flanks of each animal were clipped free of hair.

Using available information on the toxicity of the test item, a group of five male and five female rats was treated with the test item at a dose level of 2246 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight).

The appropriate amount of test item, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage. The animals were caged individually for the 24-Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were
securely in place.

REMOVAL OF TEST SUBSTANCE:
After the 24-Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.

Duration of exposure:

24 hours

Doses:

2246 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight).

No. of animals per sex per dose:

5 male and 5 female at 2246 mg/kg (2000 mg ai/kg bodyweight).

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.

After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the Draize scale (see evaluation of skin reactions).

Any other skin reactions, if present were also recorded.

Individual bodyweights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.

- Necropsy of survivors performed: yes
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

>= 2 000 mg/kg bw

Based on:

act. ingr.

Remarks on result:

other: Equivalent to >1580 mg/kg bw active acid

Mortality:

Individual mortality data are given in Table 1 (see attached background material).
There were no deaths.

Clinical signs:

other: Individual clinical observations are given in Table 1 (see attached background material). There were no signs of systemic toxicity.

Gross pathology:

Individual necropsy findings are given in Table 5 (see attached background material).
No abnormalities were noted at necropsy.

Other findings:

Dermal Reactions:
Individual dermal reactions are given in Table 2 and Table 3 (see attached background material).
There were no signs of dermal irritation.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2246 mg/kg bodyweight (equivalent to 2000 mg active ingredient/kg bodyweight, equivalent to to 1580 mg/kg bw active acid).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

1 580 mg/kg bw

Additional information

The key acute oral toxicity study, conducted according to OECD Test Guideline 423 and in compliance with GLP, identified an LD50 of 500-1000 mg/kg (equivalent to 250-500 mg/kg active acid substance, as the test material was a 50% aqueous solution) (SafePharm 2003a). In the study, a group of three fasted female rats was treated with the test item at a dose level of 2000 mg/kg bodyweight. This was followed by a second group of three fasted females treated at dose level of 300 mg/kg bw. The test item was administered orally by gavage. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. Two animals treated at a dose level of 2000 mg/kg bw were found dead or killed in extremis two hours or one day after dosing. There were no deaths at a dose level of 300 mg/kg bw. Signs of toxicity noted at a dose level of 2000 mg/kg bw were hunched posture, ataxia, decreased respiratory rate, gasping, laboured and noisy respiration, loss of righting reflex, piloerection, ptosis, pallor of the extremities and red/brown staining around the eyes. One animal treated at a dose level of 2000 mg/kg bw was comatose one hour after dosing. There were no signs of toxicity at a dose level of 300 mg/kg bw. The surviving animals showed expected gains in bodyweight over the study period. Abnormalities noted at necropsy of the animal that was found dead two hours after dosing were haemorrhagic lungs, dark liver, dark kidneys, haemorrhage of the non-glandular region of the stomach and severe haemorrhage of the gastric mucosa and small and large intestines. Abnormalities noted at necropsy of the animal that was killed in extremis one day after dosing were haemorrhage of the gastric mucosa and gaseous stomach and small and large intestines. The effects noted during the study and at necropsy were considered to be secondary to local effects of the substance based on the known pH (<2). No abnormalities were noted at necropsy of animals that were killed at the end of the study.

In a supporting acute oral toxicity study for HEBMP-xNa, conducted according to OECD Test Guideline 423 and in compliance with GLP, the LD50 value was concluded to be greater than 2246 mg/kg body weight (equivalent to 2000 mg active salt/kg body weight, equivalent to 1580 mg/kg body weight active acid) (Harlan Laboratories, 2012a). In the study, a group of three fasted females was treated with the test item at a dose level of 2246 mg/kg bodyweight (equivalent to 2000 mg active ingredient/kg bodyweight). This was followed by a further group of three fasted females at the same dose level. The test item was administered orally as a solution in distilled water. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths during the 14-day study period. There were no signs of systemic toxicity. All animals showed expected gains in bodyweight over the study period. No abnormalities were noted at necropsy.

In the acute dermal toxicity study for HEBMP-xNa, conducted according to OECD Test Guideline 402 and in compliance with GLP, the LD50 value was concluded to be greater than 2246 mg/kg bodyweight (equivalent to 2000 mg active salt/kg bodyweight, equivalent to 1580 mg/kg bw active acid, see Note 1 above) (Harlan Laboratories, 2012b). A group of ten animals (five males and five females) was given a single, 24-hour, semi‑occluded dermal application of the test item to intact skin at a dose level of 2246 mg/kg bodyweight (equivalent to 2000 mg active ingredient/kg bodyweight). Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths. There were no signs of systemic toxicity. There were no signs of dermal irritation. Animals showed expected gains in bodyweight, except for one female which showed no gain in bodyweight during the first week but expected gain in bodyweight during the second week. No abnormalities were noted at necropsy.

Justification for classification or non-classification

Discussion of classification conclusion

The classification decision is based on data for the substance itself HEBMP-H. In the absence of detailed information of the impurities in the test material used in the key data, these are considered separately for completeness. The known impurities, phosphoric acid, phosphonic acid and hydrogen chloride, are present at concentrations above 1 % in the registered substance and have classifications listed in Annex VI of Regulation (EC) No 1272/2008.

The registered substance is typically manufactured as aqueous solutions containing about 50% w/w solids hence about 50% w/w water. However, in accordance with the definition in REACH of a substance, water (as a solvent that may be separated) is not considered to be a constituent. The SIP concentration ranges are based on the substance as registered (without water); therefore, concentration ranges in the substance as sold are approximately half of the values quoted here.

Phosphonic acid, (CAS 13598-36-2, EC 237-066-7) is present at the concentration range of 0-≤5 % w/w in the registered substance HEBMP-H (equivalent to 0-2.5% of a 50% aqueous solution; no solids products are on the market). It is classified for acute oral toxicity Cat 4 according to Annex VI of CLP Regulation (EC) No 1272/2008. The impurity does not contribute to the acute toxicity of the substance and does not affect the classification conclusion for HEBMP-H based on the additivity approach.

Hydrogen chloride (CAS 7647-01-0, EC 231-595-7) is present at the concentration range of 0-≤10 % w/w (equivalent to 0-5% of a 50% aqueous solution; no solids products are on the market). It is classified for specific target organ toxicity (respiratory tract) following single exposure (STOT SE) Cat 3, H355 "May cause respiratory irritation" according to Annex VI of CLP Regulation (EC) No 1272/2008. Since, the STOT SE classification is Cat 3, hydrogen chloride does not affect the classification conclusion for HEBMP-H.

The LD50 of 500-1000 mg/kg bw derived in the acute oral toxicity study with HEBMP-H indicates that the 50% aqueous solution of the substance as tested (if sold) should be classified as acutely toxic by the oral route, Category 4, H302: “Harmful if swallowed”. This is equivalent to an LD50 of 250-500 mg/kg bw for the active acid (the substance registered), therefore the active acid should be classified as acutely toxic by the oral route, Category 3, H301: “Toxic if swallowed”.

No classification is required for acute dermal or inhalation toxicity according to Regulation (EC) No 1272/2008.